Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate?

14 Pages·2001·0.61 MB·English

85
J.Anat.(2001)199,pp.85–98,with8figures PrintedintheUnitedKingdom
Evolution of neural crest and placodes: amphioxus as a model
for the ancestral vertebrate?
LINDA Z. HOLLAND AND NICHOLAS D. HOLLAND
Scripps Institution of Oceanography, University of California San Diego, USA
(Accepted 11 April2001)
abstract
Recent studies of protochordates (ascidian tunicates and amphioxus) have given insights into possible
ancestors of 2 of the characteristic features of the vertebrate head: neural crest and placodes. The neural
crest probably evolved from cells on either side of the neural plate–epidermis boundary in a protochordate
ancestral to the vertebrates. In amphioxus, homologues of several vertebrate neural crest marker genes
(BMP2}4, Pax3} , Msx, Dll and Snail) are expressed at the edges of the neural plate and}or adjacent
nonneural ectoderm. Some of these markers are also similarly expressed in tunicates. In protochordates,
however, these cells, unlike vertebrate neural crest, neither migrate as individuals through embryonic tissues
nor differentiate into a wide spectrum of cell types. Therefore, while the protochordate ancestor of the
vertebrates probably had the beginnings of a genetic programme for neural crest formation, this programme
was augmented in the earliest vertebrates to attain definitive neural crest. Clear homologues of vertebrate
placodes are lacking in protochordates. However, both amphioxus and tunicates have ectodermal sensory
cells. In tunicates these are all primary neurons, sending axons to the central nervous system, while in
amphioxus, the ectodermal sensory cells include both primary neurons and secondary neurons lacking
axons. Comparisons of developmental gene expression suggest that the anterior ectoderm in amphioxus may
be homologous to the vertebrate olfactory placode, the only vertebrate placode with primary, not secondary,
neurons. Similarly, biochemical, morphological and gene expression data suggest that amphioxus and
tunicates also have homologues of the adenohypophysis, one of the few vertebrate structures derived from
nonneurogenic placodes. In contrast, the origin of the other vertebrate placodes is very uncertain.
Keywords:Sensorycells;protochordates;tunicates;olfactoryplacode;adenohypophysis;pituitary.
dermis,smoothmuscleoftheaorticarches,odontob-
introduction
lasts,much oftheanteriorneurocranium and dermal
Althoughamphioxusistheclosestlivinginvertebrate skeleton.
relativeofthevertebrates,itlacksmanycharacteristic Placodes are thickenings of the embryonic head
features of the vertebrate head which develop from ectoderm which, with a few exceptions such as the
2populationsofmigratoryectodermalcells,theneural adenohypophysealandlensplacodes,areneurogenic,
crest and placodes (reviewed in Gans & Northcutt, differentiatingintoneuronsaswellasothercelltypes.
1983; Northcutt 1996; Kalcheim, 2000). The neural The neurogenic placodes give rise to parts of various
crest is located at the edges of the neural plate and nerves and ganglia, the olfactory organ, parietal eye,
adjacent nonneural ectoderm along the trunk and parts of the inner ear, and the lateral line. There are
head of early vertebrate embryos. Cells from the 2majorseriesofneurogenicplacodes,thedorsolateral
neural crest migrate through the tissues of the early and ventrolateral or epibranchial. In addition, there
embryotogiverisetostructuressuchaspigmentcells, are the olfactory and profundal placodes (reviewed
cells of the adrenal medulla, cartilage, the primordia in Northcutt, 1996; Graham & Begbie, 2000). It is
of the trigeminal, facial, glossopharyngeal and vagal thought likely that the earliest jawed vertebrates had
cranialnerves,cartilageofthebranchialbars,preotic 7 pairs of cephalic dorsolateral placodes, which form
CorrespondencetoDrLindaHolland,ScrippsInstitutionofOceanography,UniversityofCalifornia,SanDiego,CA92093-0202,USA.
e-mail:lzholland!ucsd.edu
86 Linda Z. Holland and Nicholas D. Holland
much of the cranial sensory neurons including all of lamellipodia to fuse in the dorsal midline (Figs 1, 2)
thoseinvolvedinolfaction,balance,tasteandhearing (Holland et al. 1996). Subsequently, the neural plate
(Northcutt, 1996). Other placode derivatives include rolls up into the neural tube. However, unlike neural
sensoryreceptorcellsandsensoryepithelia.Although crest,thecellswithlamellipodiaremainepithelialand
the neural crest and placodes share some features, nevermigrateasindividuals.Nevertheless,cellsatthe
such as their ectodermal origin, migratory behaviour edges of the neural plate and adjacent nonneural
andabilitytoadoptmanycellfates,theydifferinthe ectoderminamphioxusexpresshomologuesofseveral
typesofcellstheygiveriseto(neuralcrestgivesriseto genes that are expressed in premigratory and mi-
morecelltypesthanplacodes),andinthesiteoforigin gratory neural crest in vertebrates, suggesting that
(neuralfoldsvscranialsurfaceectoderm).Inaddition, some of the genetic machinery for generating neural
whileneuralcrestarisesduringneurulation, placodes crestwas alreadyinplace in thecommon ancestor of
typicallyariselaterinembryonicdevelopment.There- amphioxus and the vertebrates.
fore it has been controversial as to whether neural Oneof the first genes to be expressed in nonneural
crest and placodes are ontogenetically and}or evol- ectoderminamphioxusisBMP2}4(Panopoulouetal.
utionarily related. Furthermore, since placodes do 1998). BMP2}4 homologues appear to have a very
not all share a common development (Graham & ancient role in distinguishing neural from nonneural
Begbie, 2000), there is the possibility that placodes ectoderm; in Drosophila as well as in amphioxus and
maynotallhaveacommonevolutionaryorigin.Clues vertebrates, BMP2}4 homologues are expressed in
as to the evolutionary origins of vertebrate neural nonneural ectoderm and function in distinguishing
crest and placodes have, of necessity, been sought neural from nonneural ectoderm (Francois & Bier,
amongtheinvertebratechordates(amphioxusandthe 1995;Sasaietal.1995;Wilson&Hemmati-Brivanlou,
tunicates). In recent years, comparative data from 1995; Panopoulou et al. 1998). A change in level of
electron microscopy and developmental gene expres- BMP2}4 from very high in nonneural ectoderm to
sion have suggested that invertebrate chordates, and low in neural ectoderm appears to be a key factor in
by extension the ancestral vertebrate, have popula- developmentofneuralcrest(Baker&Bronner-Fraser,
tionsofcellswithsomepropertiesofneuralcrestand 1997a,1997b;Erickson&Reedy,1998;Marchantet
placodes. The present review considers this evidence. al. 1998; Selleck et al. 1998).
Amphioxus and vertebrate Distal-less genes
(Akimenko et al. 1994; Dirksen et al. 1994; Holland
evolution of neural crest
et al. 1996) are also expressed at high levels in
Awelldevelopedneuralcrestpopulationispresentin nonneural, but not neural, ectoderm. A functional
lampreys(Horigomeetal.1999;Tomsa&Langeland, relationship between BMP2}4 signalling and expres-
1999) and gnathostomes. It is uncertain if hagfish sion of chick DLX5 has been suggested (Pera et al.
also have neural crest since their embryos are mainly 1999). Subsequently, in both vertebrates and amphi-
known from a century-old collection and there is oxus, expression of several other genes begins at this
a pressing need for a modern study of their de- boundary region. These include the snail}slug genes,
velopment. However, the existence of chordate fos- Pax3}7genes,andMsx(Mayoretal.1995;Mancilla
sils from the early Cambrian (Yunnanozoan and & Mayor, 1996; Conway et al. 1997; Maeda et al.
Haikouella) with apparent neural-crest derived struc- 1997; Jiang et al. 1998; LaBonne & Bronner-Fraser
tures (pharyngeal denticles and pharyngeal skeletons 1998; Langeland et al. 1998; Sefton et al. 1998;
resemblingthestripedmucocartilageofthebranchial Sharmanetal.1999;Hollandetal.1999).Thesegenes
bars in lamprey ammocoete larvae), suggests that are all strongly expressed in the lateral edges of the
neural crest arose very early in vertebrate evolution neural plate. In amphioxus, later expression of
(Chen et al. 1999; Holland & Chen, 2001). AmphiPax3}7, like vertebrate Pax3 becomes restric-
The invertebrate chordates apparently lack defini- ted to dorsal domains in the neural tube (Holland
tiveneuralcrest.Onemarkerofmigratingneuralcrest et al. 1999). Similarly, AmphiMsx continues to be
in some vertebrates, the antibody HNK1, does not expressed in dorsal cells of neural tube in the mid-
recognize any cells in amphioxus embryos (our neurula (Sharman et al. 1999). In the early neurula,
unpublished data). Even so, in both amphioxus and transcriptsalsoappearincellsinthedorsalportionof
tunicates, cells at the edges of the neural plate and the somites. Although Sharman et al. (1999) specu-
adjacentnonneuralectodermsharesomepropertiesof lated that these cells might be evidence of wandering
neural crest. In amphioxus, the nonneural ectoderm neural crest, an alternative explanation is that the
detachesfromtheneuralplateandmigratesoveritvia gene is being expressed in somitic cells as noted for
Evolution of chordate ectodermal sensory cells 87
Fig. 1. Diagram of neurulation in amphioxus. (A) Late gastrula. The ectoderm (ec) is flattening dorsally to form the neural plate. me,
mesendoderm.(B)Early neurula.The nonneural ectoderm has detached(*) from the edgesof the neural plate neural plate (np)and is
beginningtomigrateoverthedorsalsideoftheembryo.(C)Theneuralplateisbeginningtoroundupastheoverlyingectodermmigrates
overit.(D)Mid-neurula.Thenonneural ectodermhas fused (*) dorsally over the forming neural tube.Presomitic grooves are forming
dorsolaterally as outpocketings from the embryonic gut (arrows). The notochord is also forming axially as an outpocketing from the
embryonicgut(arrowhead).(E)Lateneurula.Thenervecord(nc)hasformed,andthesomites(so)andnotochord(no)havepinchedoff
fromtheembryonicgut(en).
Fig.2. SEMof neurulationin amphioxus.(A)Dorsal viewof an8-hneurulawiththe anterior toward the bottomleft.Thenonneural
ectodermisbeginningtomoveovertheneuralplate(np).Mostoftheectodermalcellshaveasinglecilium(arrows).Bar,20lm.(B)Higher
magnificationofcellsatleadingedgeofnonneuralectodermadvancingoverneuralplate(topleft),withlamellipodia(arrowhead)extended
onthesurfaceoftheneuralplate(np).Bar,5lm.FromHollandetal.(1996).
XenopusbyMaedaetal.(1997)andHouzelsteinetal. expression of Pax3}7 and Msx in the edges of the
(2000). neural plate has been shown (Dorsky et al. 1997,
In vertebrates, induction of neural crest also 2000; LaBonne & Bronner-Fraser, 1998; Bang et al.
involvesWntgenes.InXenopus,aroleformesodermal 1999).Inamphioxus,Wnt8isexpressedlikevertebrate
expression of Wnt8 in induction of neural crest and Wnt8 in the paraxial mesoderm, and it may similarly
88 Linda Z. Holland and Nicholas D. Holland
be involved in the induction of the expression of 1998). While expression is lost from the descendants
Pax3}7 and msx in cells at the edges of the neural of these cells by the onset of neurulation, a lacZ
plate (Schubert et al. 2000). In addition, Xenopus reporter construct demonstrated Ci-sna expression in
Wnt7Bhasrestrictedexpressioninthedorsalportion lateral ependymal cells of the tail nerve cord. The
oftheneuraltube(Chang&Hemati-Brivanlou,1998), patterns of Ci-sna and HrPax-37 appear to overlap
and injection of Wnt7B, together with the neural but are not identical. Both genes are expressed in
inducer noggin, induces expression of neural crest progenitorsofthedorsalcerebralvesicle,butHrPax-
markers such as Slug and twist. In contrast, while 37 is expressed in the progenitors of the dorsal, not
amphioxusWnt7Bisalsoexpressedintheneuraltube, lateral, ependymal cells. Similarly, an ascidian Msx
itisexpressedatalldorsoventrallevels(Schubertetal. geneisexpressedintheneuralfoldaswellasinother
2000). While it could be one factor in induction of tissues(Maetal.1996).However,expressionofWnt7
Snail expression at the edges of the neural plate, is restricted to dorsal and ventral ependymal cells in
restriction of Wnt7B expression to the edges of the thetailnervecord(Sasakura&Makabe,2000).Thus,
neural plate may have occurred together with the although the nerve cord of ascidian tunicates has
evolution of definitive neural crest. relatively few cells, and the tail nerve cord lacks cell
Ascidianhomologuesofsomeoftheseneural crest bodies, cells at the edges of the neural plate and
markers are also expressed in the edges of the neural adjacent nonneural ectoderm do express some of the
plate and}or adjacent nonneural ectoderm. Excep- gene markers of neural crest. Thus, the cells at the
tions are BMP2}4 and distal-less. Ascidian embryos edge of the neural plate and adjacent nonneural
have early determination of cell fates and a reduced ectoderm in the ancestral chordate had evolved at
number of embryonic cells and, perhaps as a result, least a few properties that characterize neural crest.
BMP2}4appearstohavelostaroleindistinguishing To date, the rule that markers of premigratory
neural from nonneural ectoderm. Although ascidian neuralcrestaresimilarlyexpressedinamphioxusand
BMP2}4isexpressedinsomecellsattheedgesofthe vertebrates has one important exception. Twist is
neural plate, it is not expressed in the nonneural expressed in neural crest in Xenopus(Hopwood et al.
ectoderm during gastrulation (Miya et al. 1997). 1989).Inamphioxus,thehomologueoftwist,Bbtwist
Moreover, none of the 3 ascidian distal-less genes is (so called after the species, Branchiostoma belcheri
expressedincellsattheboundaryofneuralplateand from which it was cloned), is expressed only in the
nonneural ectoderm (Caracciolo et al. 2000). Genes mesoderm and endoderm (Yasui et al. 1998a). It is
which are expressed at the edge of the neural plate likelythatotheramphioxusandtunicatehomologues
and}or adjacent nonneural ectoderm include homol- of vertebrate neural crest markers will be found that
oguesofPax3}7,SnailandMsx.Initialexpressionof are not expressed at the edges of the neural plate. A
these genes occurs relatively early in development, betterunderstandingofwhichamphioxushomologues
perhapsbecauseoftheveryearlydeterminationofcell ofvertebrateneuralcrestmarkersareexpressedatthe
fates. Since ascidian embryos have few cells, these edge of the neural plate and which are not should
genes are also expressed in relatively few cells. In the facilitate an understanding of the genetic controls of
late gastrula of Halocynthia roretzi, HrPax-37, the theabilityofneuralcresttomigratethroughtissuesas
homologueofvertebratePax3andPax7,isexpressed single cells.
oneachsideoftheembryoin3dorsalcellswhichwill A start at determining how the evolution of
form part of the neural plate, although 2 will also regulatoryregionsofgenesisrelatedtotheevolution
form muscle (Wada et al. 1996). Later, HrPax-37 of neural crest comes from cross-species analysis of
expressiondecreasesintheneuralplateandincreases reporter constructs. Manzanares et al. (2001) have
in adjacent epidermal cells. Although somewhat analysed expression in vertebrates of a series of
different from that of vertebrate or amphioxus reporter constructs of regulatory regions of the
Pax3}7,thisexpressiondoessuggestaroleforHrPax- amphioxus 3« Hox genes, AmphiHox1, AmphiHox2,
37 in patterning the edges of the neural plate and and AmphiHox3. In vertebrates, these genes are
adjacentnonneuralectoderm.Inaddition,overexpres- involvedinanterior–posteriorpatterningoftheneural
sion of HrPax3}7 induces expression of some dorsal tube.Theyarealsoexpressedincertainpopulationsof
genes (Wada et al. 1997). Like vertebrate and neural crest cells. However, their initial expression is
amphioxus Snail}slug genes, Ci-Sna, the ascidian later than that of the premigratory neural crest
homologue, is expressed in cells at the lateral neural markers discussed above, and, therefore, these Hox
plate border as well as in muscle and trunk mes- genes probably act downstream of these markers.
enchyme precursors (Corbo et al. 1997; Erives et al. Manzanares et al. (2001) found that some regulatory
Evolution of chordate ectodermal sensory cells 89
elements(e.g.bindingsitesfornuclearreceptorssuch nonneurogenicplacodesgiverisetosuchstructuresas
astheretinoicacidreceptor)wereconservedbetween the adenohypophysis, teeth, feathers, hair and lens.
amphioxus and vertebrate Hox genes. However, Theneurogenicplacodesgiverisetoawidevarietyof
binding sites for Krox20 and kreisler present in the mechano-andchemosensorystructures,includingthe
vertebrateHoxgeneswereabsentfromtheamphioxus olfactory epithelium of the nose, parts of cranial
counterparts. Interestingly, one amphioxus reporter ganglia, lateral line, sensory cells of the inner ear,
constructdirectedexpressiontobothneuralcrestand some of the GNRH-containing neurons of the
placodes in vertebrates. This result indicates that at pituitary. Lateral lines are present only in agnathans
leastsomeregulatoryelementsnecessaryfordirecting andinanamniotes.Exceptforthesensorycellsinthe
Hoxexpressioninneuralcresthadevolvedbeforethe olfactoryplacode,whichareprimaryneurons,sending
neural crest precursors had developed the ability to axons towards the central nervous system, all of the
migrate. However, the expression of Hox genes in neuronal cells in neurogenic placodes are secondary
migrating neural crest cells is probably unrelated to neurons, lacking axons.
their acquisition of the ability to migrate as individ-
uals. In mice mutant for some of the 3« Hox genes,
evolution of neurogenic placodes
neural crest generally forms and migrates, although
the identity of structures made by neural crest is Amphioxus and the tunicates have numerous ec-
typicallyaltered(Condie&Capecchi,1993;Manley& todermal sensory cells (Fig. 3). However, none of
Capecchi, 1997, 1998). The function these specific theseismorphologicallyidenticaltoanyofthesensory
regulatory elements play in amphioxus embryology cells of vertebrate placodes, and there has thus been
awaits the development of techniques for expression considerable argument concerning the evolutionary
ofreporterconstructsinamphioxusembryos.Never- relationship, if any, between the ectodermal sensory
theless, taken together, these results plus the ex- cells of protochordates and vertebrate placodes. The
pression of neural crest markers in amphioxus and most characteristic ectodermal sensory cells in verte-
ascidians suggest that substantial parts of the gene brates are the mechanosensory cells in vertebrate
networks specifying neural crest were in place in the lateral lines. These are organised into neuromasts,
common ancestor of amphioxus and the vertebrates. whichconsistofciliatedhaircellsandsupportingcells
with their tips embedded in a gelatinous cupule. The
‘hairs’ on a hair cell consist of one immobile
placodes
kinocilium and numerous motile stereovilli, which
Placodes are thickenings of the head ectoderm in have also been called stereocilia (Fig. 4). The
embryonic vertebrates. Two types of placodes— kinocilium is a typical cilium with a basal body and
neurogenic and nonneurogenic—are recognised. The core of microtubules. However, the stereovilli are
Fig. 3. Chordate epidermal sensory neurons, excluding photoreceptors and sensory neurons with endings in mesodermal structures.
TunicateshavetypeIsensoryneurons(1),whichsendanaxonalprocesstotheCNS.TheepidermisofamphioxushasbothtypeI(1)and
typeII(2)sensoryneurons.ThetypeIIcellssynapsewithasensoryneuronthatsendsanaxontotheCNS.3*,Retziusbipolarcell.The
cellbodyisintheCNS.Itisequivocalwhethertheprocessofthesecellsterminateintheepidermis.Asimilarneuron,theRohon-Beard
cellistransientlypresentinearlyembryosofanamniotes.Invertebrates,typeIsensorycells(4)arepresentintheolfactoryplacode.Other
ectodermalsensorycellsaresecondaryneurons(5).Inadultvertebrates,theRohon-Beardcellisreplacedbydorsalrootganglia(6).
90 Linda Z. Holland and Nicholas D. Holland
similarity with the cupule of neuromasts. Like
vertebrateneuromasts,theciliaoftheascidiancupular
organs are embedded in extracellular material (Bone
& Ryan, 1978). In ascidians, however, this material
appears to be simply part of the test that covers the
entire organism. In addition, the sensory cells in the
ascidian cupular organs and vertebrate neuromasts
areratherdifferent.Theascidiancupularcellshave a
single,longcilium,surroundedbyfromzerotomany
microvilli.However,theseciliaandmicrovillilackthe
characteristic specialisations of stereovilli and kino-
cilia (Fig. 4). Moreover, these ascidian cells have
axons (Bone & Ryan, 1978). Nevertheless, the ex-
pression of the ascidian homologues of Pax2}5}8 in
theascidianatrialprimordiahasbeentakeninsupport
oftheirhomologywiththeoticplacodeofvertebrates
(Wadaetal.1998).However,expressionofamphioxus
Pax2}5}8 suggests an alternative hypothesis. Amphi-
Pax2}5}8isnot expressed in anyectodermal sensory
cells.Itis,however,expressedintheforminggillslits
at the point where the ectoderm and endoderm will
fuse.Inascidians,theprimordiaoftheadultoraland
atrial siphons, where the cupular organs develop, are
formed by invaginations of ectoderm (Katz, 1983).
Thus it could be that, similar to AmphiPax2}5}8,
ascidian Pax2}5}8 is expressed in ectoderm that will
Fig.4. Comparison of asensory cell in the cupularorganof the invaginate and form the outer part of the gill slits
ascidian Ciona (A) and a sensory cell (hair cell) from the fish by fusion with endoderm. If so, the ascidian atrial
lateralissystem(B).(A)Inthetunicatecupularorgan,thesensory
primordiamaynotbehomologoustooticvesicles,but
cell is a primary neuron sending an axon (ax) to the CNS. The
sensory cilium (sc) is within a deep indentation of the cell and is totheouterpartofgillslits(Kozmiketal.1999).The
surroundedbyacollarofshortmicrovilli.(B)Thevertebratehair homology of ascidian cupular organs and vertebrate
cellisasecondaryneuron,synapsingwithefferent(ef)andafferent
neuromastshasalsobeendoubtedbecauseinhagfish,
(af)fibresofthecranialnerves.Thereisasingleapicalkinocilium
(kc) and an array of stereovilli (sv) filled with fasicles of actin cupulae are absent in the lateral line grooves and the
filaments.(ModifiedfromBone&Ryan,1978.) cristaeofthesemicircularcanals(Braun&Northcutt,
1997). Cupulae probably also are absent in the
specialised microvilli. They are long (upwards of neuromasts of lampreys (Braun & Northcutt 1997).
5lm),packedwithaparallelarrayofmicrofilaments, Thus they may have evolved independently in asci-
andtheirtipsarelinkedtogetherbyfineextracellular diansandhighervertebrates.Thisindependentorigin
threads.Whenmechanicallystimulated,thestereovilli is supported by the lack of any similar structure in
movetogether,inducingreceptorpotentialsacrossthe amphioxus and by the lack of expression of charac-
cell membrane. Hair cells, which are secondary teristicoticplacodegeneticmarkers(suchasSox3and
neurons lacking an axonal process, also occur in the Pax2}5}8)intheectodermoppositethemiddleofthe
vertebrate inner ear. amphioxushindbrainwhereonewouldexpectanotic
placode to be (Kozmik et al. 1999).
Ascidianshaveafewadditionalectodermalsensory
Ectodermal sensory cells in tunicates
cells. There are ciliated sensory cells on the rostral
Amphioxus has nothing that resembles a vertebrate papillae, which attach the larva to the substratum at
neuromast. However, in the developing pharynx of metamorphosis. At the base of each papilla there are
adultascidians,therearetheso-calledcupularorgans, several uniciliate sensory cells with axonal processes
whichhavebeensuggestedaspossiblehomologuesof (Torrence & Cloney 1983; Gianguzza & Dolcema-
neuromasts in the inner ear (Bone & Ryan, 1978; scolo 1994, 1997; Sotgia et al. 1998). It has been
Wada et al. 1998). The term ‘cupular’ was evidently suggested that the ascidian adhesive papillae are
used by Bone & Ryan (1978) to point out the relatedtotheamphibiancementgland(Pennatietal.
Evolution of chordate ectodermal sensory cells 91
Fig.5. TEMofectodermalsensorycellsofamphioxus.(A)TypeIsensorycellshowingaboutthelowerhalfofthecilium,whichhasatypical
9›2arrayofmicrotubules.(B)TypeIIsensorycell.Theshortciliumiscompletelyfilledbyanarrayofabout20–30axialmicrotubules.
Theciliumissurroundedbyanextendedmicrovillarcollarstuddedwithshortbranches.(FromLacalli&Hou,1999.)
1998). These authors found that the ascidian hom-
Ectodermal sensory cells in amphioxus
ologueofanamphibiangeneXAG,whichisexpressed
in the cement gland, is expressed in the apex of the Compared to tunicates, amphioxus has far more
adhesive papillae. Similar sensory cells have been larvalectodermalsensorycells,manyofwhichpersist
described in the larval tail of tunicates (Torrence & in the adult. There are several types of ciliated
Cloney, 1982; Crowther & Whittaker, 1994). epidermal cells with distinctive morphology (Welsch,
Someectodermalsensoryorgansarealsopresentin 1968; Bone & Best, 1978; Baatrup, 1981; Stokes &
other tunicates. There is asense organ just ventral to Holland, 1995; Lacalli & Hou, 1999). Type I cells
the mouth in the appendicularian Oikopleura dioica have a collar of short microvilli surrounding the
(Bollner etal.1986). This sense organ includes about cilium (Fig. 5A). These cells appear after 48h of
30 uniciliate cells, and it has been suggested that it is developmentalongthesidesofthemiddlethirdofthe
chemosensory, perhaps evolutionarily related to the larva, becoming more widespread as development
vertebrate olfactory apparatus (Bollner et al. 1986). continues (Schulte & Riehl, 1977; Stokes & Holland,
However,aswithectodermalsensoryorgansofother 1995).Theybecomeparticularlyconcentratedontheir
invertebrate deuterostomes, no direct tests of the rostral epidermis. Two subtypes of type I cells have
exact sensory modality of this receptor have been beenrecognised.Bothhavealongcilium,upto10lm
made. inlength,whichisnotrecessed.However,thetypeIa
92 Linda Z. Holland and Nicholas D. Holland
Fig.6. Diagramofa12–14damphioxuslarvashowingseveralminortypesofectodermalsensorycells.Themouth,whichopensonthe
left,issurroundedbyoralspinecells,whicharesecondarysensorycellssynapsingpredominantlywithdendrite-likecellprocessesonintrinsic
neurons.Theoralspinesarisefromclustersofflask-shapeduniciliatecell.Themouthissurroundedbytheoralnerve.Theorgansofde
Quatrefagesareindicatedbyarrows.Theysendaxonstotherostralnerveandthefirstdorsalnerve.Theciliatedpit(p),whichjoinswith
Hatschek’sdiverticulumtoformHatschek’spitintheadult,isanteriortothemouth.Cellsofthetherostralpapilla(rp)andoralpapilla
(op)havelongcilia.(ModifiedfromLacallietal.1999.)
cell is surrounded by 2 morphological types of arecellswithciliaryspines.Theaxonemehasanaxial
microvilli, thick ones in an inner ring, and more fibrouscorewithhorizontalplatesatregularintervals
slender ones in an outer ring (Lacalli & Hou, 1999). along the whole length of the cilium. Lacalli & Hou
All the microvilli around the cilium on type Ib cells (1999) suggested that these are mechanoreceptors.
are slender. Type Ib cells are primary neurons with Around the mouth are oral spine cells, which are
axonsthatprojectthroughthefirstandseconddorsal secondary sense cells with immobile cilia that project
sensory nerves into the anterior part of the CNS, the across the mouth (Fig. 6). The oral spines act to
cerebralvesicle(Fritzsch,1996).WhethertypeIacells initiate a larval ‘cough’ response that clears the
have axons or not is less certain. mouth of debris. The oral spine cells synapse with
Late in larval life, type II sensory cells appear neurites of some peripheral interneurons, which in
(Schulte&Riel,1977;Stokes&Holland,1995).These turn synapse with peripheral neurons having long
cells have a cilium 3lm long with a swollen tip (Fig. axons, which are thought to be sensory cells, trans-
5B).Theciliumissurroundedbyacollarstuddedwith mitting signals to the nerve cord. These peripheral
microvilli.TypeIIcellsaresecondaryneurons,lacking interneurons resemble the Merkel-like accessory cells
axons,synapsingviashortcellprocesseswithsensory ofthevertebratetastebuds.ThusLacallietal.(1999)
cells that send axons to the CNS (Lacalli & Hou, suggested that the oral spine cells might be hom-
1999). After metamorphosis, the preoral cirri have ologoustovertebratetastebuds,which,atleastinthe
type I and possibly type II cells as well (Schulte & axolotl,areneitherplacodallynorneuralcrest-derived
Riehl,1977;Bone&Best,1978).Whileitisgenerally (BarlowandNorthcutt,1995).Theciliarytuftororal
agreed that both type I and type II cells are sensory, papilla (Andersson & Olsson, 1989; Stokes & Hol-
it is a matter of controversy whether they are land, 1995; Lacalli et al. 1999) is located anteriorly
mechano- or chemosensory (Schulte & Riehl, 1977; and slightly ventral to the mouth. The cilia of these
Bone & Best, 1978; Baatrup, 1981; Lacalli & Hou, cells project forward during feeding and beat ir-
1999). Even though amphioxus is known to respond regularly and slowly. A sensory role has been
to chemical and touch stimuli and chemical change, suggested for these cells (Gilmour, 1996), but Lacalli
no direct tests of the function of individual cell types et al. (1999) suggested that they may be ciliary
have been done (Parker 1908). effectors rather than sensory cells since they do not
There are several minor types of sensory cells seem to form synapses with the oral nerve.
(Fig.6).Theseincludecellsontherostralsurfacewith
cilia recessed into irregular pits with scattered micro-
Possible homologues between amphioxus ectodermal
villi. The ventral pit cells, which are located on the
sensory cells and vertebrate placodes
metapleural folds, also have recessed cilia but lack
microvilli (Stokes & Holland, 1995; Lacalli & Hou, Whether the ancestral ectodermal sensory cell of
1999).Intheciliaofthesecells,asinthetypeIIcells, vertebrateswas aprimary neuron,sending an axonal
the axoneme is replaced by an array of parallel processtothecentralnervoussystem,orasecondary
microtubules. On the right side of the rostrum there neuron, lacking an axon, is controversial; see, for
Evolution of chordate ectodermal sensory cells 93
example, the discussion in Fritzsch (1996). In verte- early in development, and type II cells, which appear
brates, sensory cells in the olfactory placode are muchlater.Bone&Best(1978)suggestedamechano-
primary neurons. Other placodal sensory cells are receptoryfunctionforthetypeIIcellsofamphioxus.
secondaryneurons.Ithasgenerallybeenthoughtthat Baatrup(1981)couldnotdeduceafunctionfrom the
theancestralvertebratesensorycell had anaxon and morphology of these cells, while Lacalli and Hou
was,therefore,similartothetypeIcellsofamphioxus. (1999) suggested that the type Ia cells were likely
However,Lacalli&Hou(1999)suggestedthatiftype mechanoreceptors and the type II cells were more
II cells are chemosensory, they could be closer than likely to be chemosensory.
type I cells to vertebrate chemoreceptors. Braun & Sharman et al. (1999) suggested that rostral ex-
Northcutt(1997)pointed outthat the ciliated cells in pression of AmphiMsx might mark the future cor-
the ascidian cupulae, the lateral line cells of hagfish puscles of de Quatrefages, which have no clear
and both the type I and type II sensory cells in counterpartinotherchordates(Fig.6).Thecorpuscles
amphioxus are similar in having a central cilium of de Quatrefages are specialised organs, presumably
surrounded by a collar of microvilli. Thus, in sensory,locatedinthesubepidermalconnectivetissue
agreement with Jørgensen (1989), they maintained attherostralendof latelarvalandadult amphioxus.
thattheancestralchordatesensorycellmayhavehad They consist of between 1 and 4 nerve cells with
asimilarmorphology.However,theydidnotaddress axons,eachwith2cilia,surroundedbyupto7sheath
the question of whether the ancestral vertebrate cells. The cilia extend into a protrusion of the basal
sensory cell had an axon or not. lamina which surrounds each corpuscle. The axonal
Comparisons of developmental gene expression process contributes to the rostral nerve (Baatrup,
between vertebrates and amphioxus can suggest 1982;Fritzsch,1996;Demskietal.1996;Yasuietal.
possible homologies. Recently, several vertebrate 1998b). It is usually presumed that the corpuscles of
placodes have been found to express particular de Quatrefages are touch or pressure receptors
constellations of transcription factors during devel- (Baatrup, 1982; Ruppert, 1997), although it has
opment.Theolfactoryepithelium,whichgivesriseto recently been suggested that they might instead be
the olfactory placode is among the first to express chemosensory(Baker&Bronner-Fraser,1997b).This
particular transcription factors. The most specific does not seem likely because of their subepidermal
marker of the olfactory epithelium is Pax-6, which location. Nevertheless, Toresson et al. (1998) noted
amongtheotherplacodesisexpressedonlyinthelens expression of amphioxus BF-1, which is expressed
placode (Pu$schel et al. 1992). Others, such as several in the vertebrate olfactory placode, in cells where
Six genes, eya, frizzled 2, frizzled 7, Msx-1, Msx-2, the rostral nerve enters the central nervous system
Sprouty4, are less specific markers of the olfactory in support of this idea. However, expression of
placode, since they are expressed in a few other AmphiBf-1intheanteriorpartoftheamphioxusCNS
placodes aswell (Metscheretal.1997;deMaximy et couldsimplybeevolutionarilyrelatedtoitsexpression
al. 1999; Esteve and Bovolenta, 1999; Lo!pez-Rı!os et in the anterior part of the vertebrate diencephalon.
al. 1999; Kobayashi et al. 2000; Pandur & Moody, A final type of sensory cell in amphioxus, the
2000; Stark et al. 2000). Other genes (Sox2, Sox3, Retzius bipolar cell, deserves mention, although its
Neurogenin1 and 2, Delta-1, MyT-1, NeuroD) are cellbodyisnotintheectodermbutwithinthecentral
expressedinmost,ifnotall,placodes(Collignonetal. nervoussystem(Fig.3).However,itisanectodermal
1996;Mizusekietal.1998;Begbieetal.1999;Ko$ster sensorycellinthatitprobablysendsanaxonalprocess
etal.2000;SchlosserandNorthcutt,2000).Theonly to the ectoderm. The cell bodies of these cells are
amphioxushomologuesofthesegenesthathavebeen located in 2 columns in the dorsolateral part of the
cloned are Pax-6, Neurogenin, Msx and Sox1}2}3 nervecord.Theprocessesofthesecellsappeartoform
(Glardonetal.1998;Sharmanetal.1999;Hollandet mostofthenerve-fibersthatterminateintheepidermis
al. 2000a,b). Expression of AmphiSox1}2}3 is re- (Bone, 1959). However, Lacalli & Hou (1999) were
strictedtothecentralnervoussystem.However,both unable with transmission electron microscopy to find
AmphiPax-6 and AmphiNeurogenin are broadly ex- any small neurites travelling to the body surface. It
pressed in the rostral ectoderm of early larvae, while has been suggested that these cells are similar to
AmphiMsxisexpressedinapatchofrostralectoderm Rohon-Beardcellsinlampreys,teleostfishandlarval
on each side of the larva. Thus amphioxus may have amphibians (Demski et al. 1996). If so, however, the
a homologue of the olfactory epithelium. In amphi- Retzius bipolar cells apparently do not, like Rohon-
oxus,therostralendofthelatelarvaandadultisrich Beard, cells express islet. The only islet-expressing
in sensory cells:both type I cells, which appear quite cells in the dorsal part of the developing amphioxus
94 Linda Z. Holland and Nicholas D. Holland
nerve cord are located at the level of somite 5 and 6,
and are not distributed along the length of the nerve
cord like the Retzius bipolar cells (Jackman et al.
2000).
nonneurogenic placodes
Homology of the vertebrate adenohypophysis with
ascidian and amphioxus structures
With the exception of the adenohypophysis, homol-
ogues of the nonneurogenic placodes (e.g. placodes
givingrisetotheteeth,hairfolliclesandlens),appear
to be lacking in invertebrate chordates. Several lines
ofevidencepointtoHatschek’spitinadultamphioxus
asthehomologueoftheadenohypophysis.Hatschek’s
pit develops from fusion of the ciliated pit, located
anteriortothelarvalmouth,andtheleftanteriorgut Fig.8. Crosssectionof the dorsalregion of anadultamphioxus
diverticulum (also called Hatschek’s diverticulum) (Branchiostoma belcheri). A ventral extension (arrowhead) of the
nervecordgoesaroundthenotochordtoapproachHatschek’spit,
(Hatschek,1884)(Figs7,8).Hatschek’spitwasfound
the probable homologue of the vertebrate adenohypophysis
(arrow).(ModifiedfromGorbman1999.)
to be immunoreactive for GNRH (Fang et al. 1999)
andforPit-1protein,atranscriptionfactorexpressed
intheanteriorpituitaryinvertebratesbyCandiani&
Pestarino(1998),whosuggestedthatHatschek’spitis
homologous to the adenohypophysis. In addition, in
vertebrates,Pax6andislet1areexpressedinepithelial
cells of the developing adenohypophysis (Walther &
Gruss, 1991; Inoue et al. 1994; Grindley et al. 1995;
Terzic & Saraga-Babic, 1999). Similarly, amphioxus
Pax6 and islet homologues are expressed in Hat-
schek’sdiverticulum(amesodermalderivative)inthe
earlyamphioxuslarva(Glardonetal.1998;Jackman
et al. 2000).
In ascidians, there are some indications that the
Fig. 7. Development of Hatschek’s pit, the probable amphioxus neurohypophyseal duct is homologous to the adeno-
homologue of the vertebrate adenohypophysis. A–C are dorsal
hypophysis. During formation of the adult ascidian,
views; D–F are views from the left side. (A) At the mid-neurula
Hatschek’sleft(hld)andright(hrd)diverticulaformasoutpocket- the ectodermal neurohypophyseal duct fuses with an
ingsoftheendodermattheanteriorendofthepharynx(ph).(B) evagination of the pharynx to form the neural gland
AtthelateneurulaHatschek’sleft(hld)andright(hrd)diverticula
rudiment. The neural gland, like the vertebrate
pinchofffromthepharynx(ph).Themouthbeginstoformonthe
left side by fusion of the ectoderm and endoderm. (C,D) In the adenohypophysis, has immunoreactivity for GnRH
early larva Hatschek’s left diverticulum fuses with the preoral (Mackie,1995;Powelletal.1996;Tsutsuietal.1998),
ciliated pit (pocp), and the mouth opens. (E) As metamorphosis prolactin (Pestarino, 1984a; Terakado et al. 1997)
getsunderway,theanterioredgeofthemouthrotatesintotheplane
andothercharacteristichormones(Changetal.1982;
ofthepage.Thesmalltriangleonthepreoralciliatedpitindicates
theregiondestinedtobecomeHatschek’spitaftermetamorphosis. Pestarino, 1984b, 1985a–c; Terakado et al. 1997)
(F) Later in metamorphosis the epidermal region anterior to the supporting homology with the adenohypophysis.
mouthrotates(openarrows),thusmovingthepreoralciliatedpit
Consequently,notingaroleformigratoryectodermal
fromtheleftsidetotheventralside,whereitbecomestheprebuccal
cavity.(G).AftermetamorphosisHatschek’spit(hp)islocatedin cellsinneurogenesisofascidians,Mannietal.(1999),
the roof of the prebuccal cavity (pbc), just anterior to the velar revivedtheideaofhomologybetweentheneurohypo-
mouth(vmo).Insomespeciesaprojectionfromthenervecord(nc)
physealductofascidiansandthepituitaryplacodesof
extendsaroundtheleftsideofthenotochord(no)andapproaches,
vertebrates. While this homology has been cast into
butdoesnottouch,Hatschek’spit.(FromHollandetal.1998.)